Colony stimulating factor refers to a family of lumphokines which induce progenitor cells found in the bone marrow, to differentiate into different types of mature blood cells. The particular type of mature blood cell that results from a progenitor cell depends on the type of colony stimulating factor present. For instance, erythropoietin causes progenitor cells to mature into erythrocytes while thrombopoietin is thought to drive progenitor cells along the thrombocytic pathway. Similarly, granulocyte-macrophage colony formation is dependent on the presence of a granulocyte-macrophage colony stimulating factor (hereinafter referred to as "CSF"). The present invention concerns the production of CSF from malignant cells, the resolution of CSF into distinct species, and the purification of one of the species (CSF-2.alpha.) to homogeneity.
Researchers have reported the detection and production of CSF from a variety of different sources. CSF has been detected in serum and urine. Robinson et al., 69 J. Cell. Physiol. 83-92 (1967); Stanley et al., 79 J. Lab. Clin. Med. 657-668 (1972). CSF also has been extracted from substantially all of the organs of the body. Sheridan and Stanley, 78 J. Cell. Physiol. 451-459 (1971). Several researchers have reported the production of CSF from both human and monkey peripheral blood cells which appear to be macrophages or monocytes. Moore and Williams, 80 J. Cell. Physiol. 195-206 (1972); Golde and Kline, 51 J. Clin. Invest. 2981-2983 (1972); Moore et al., 50 J. Natl. Cancer Inst. 591-601 (1973). In the past, CSF also has been produced by T and B cells stimulated with serum and an appropriate plant mitogen. Parker and Metcalf, 112 J. Immunol. 502-510 (1974). CSF also has been produced by culturing T-cell hybridomas in the presence of a T-cell mitogen. Clark-Lewis and Schrader, 128 J. Immunol. 168-174 (1982).
Although the factors identified by the above researchers have been reported to be CSF, heretofore CSF has not been purified to homogeneity. As a result, relatively little is known about the specific functions and activities of the various subspecies of CSF.
Burgess et al., 185 Bilochem. J. 301-314 (1980), discussed the simultaneous production of granulocyte/macrophage (CSF), eosinophil, megakarocyte and erythroid regulatory factors by activating murine spleen cells with pokeweed mitogen in the presence of human serum. Burgess et al. reported "partial purification" of these factors by a procedure involving ammonium sulfate precipitation/dialysis, gel filtration chromatography (Sephadex G-500), ionic exchange chromatography (DEAE cellulose), concanavalin A sepharose chromatography, flat bed gel isoelectric focusing, hydrophobic chromatography (phenyl-Sepharose). No appreciable separation of the factors thus achieved by gel filtration, concanavalin A sepharose chromatography or ammonium sulfate precipitation. Although, partial purification was achieved by isoelectric focusing, none of the CSF species, so separated could be shown to be homogeneous.
Williams et al., 11 Differentiation 59-63 (1978), reported the deriving of colony stimulating activities from a conditioned medium of a myelomonocyte leukemic cell line designated as WEHI-3. The WEHI-3 conditioned medium was found to stimulate formation of granulocyte, macrophage and megakaryocyte colonies. However, when the WEHI-3 condition medium was subjected to DEAE-Sephadex A-25 column chromatography, it appeared that separate activities were fractionated, one stimulating macrophage colony formation while the other stimulating granulocyte colony formation, suggesting that granulocyte and macrophage development may be dependent upon separate regulators. See also, Horiuchi and Ichikawa, 110 Exp. Cell Res. 79 (1977).
Clark-Lewis and Schrader, supra, reported the production of CSF, T-cell growth factor (TCGF) (also known as interleukin-2) and T-cell replacing factor (TFR) from a T-cell hybridoma stimulated with concanavalin-A. Techniques used for fractionating and purifying the three factors included ammonium sulfate precipitation, gel filtration, heat treatment and hydrophobic chromatography. The factors could not be separated from each other by ammonium sulfate precipitation. The CSF product was found to have a molecular weight of from 25,000 to 30,000 by gel filtration in phosphatebuffered saline (PBS) and of about 23,000 by gel filtration in guanidine hydrochloride.
Accordingly, the principal objects of the present invention are to identify cell lines and clones thereof which are potent producers of CSF and to purify the CSF to homogenity thereby to resolve CSF into identifiable, distinct species.
Another object of the present invention is to purify crude preparations of CSF to homogeneity by reverse phase, high performance liquid chromatography techniques.
A further object of the present invention is to ascertain, at least in part, the amino acid sequence of CSF.